Project Summary This proposal describes a five-year program to investigate the roles of paired-like homeodomain 2 (Pitx2) signaling during cellular injury response and fibrotic scar formation after myocardial infarction (MI). MI accounts for millions of deaths worldwide annually. Therefore, developing an effective regenerative therapy is one of the major goals of modern cardiovascular biology. Pitx2, when overexpressed in cardiomyocytes, is capable of partially repairing myocardium in a mouse MI model. Pitx2 function is induced and promoted by the upstream nuclear factor erythroid 2 like 2 (Nrf2), a known regulator of redox balance. Unlike most of the regeneration- promoting factors that induce cell cycle reentry in cardiomyocytes, the Pitx2 signaling only has a mild effect on cardiomyocyte proliferation. Instead, Pitx2 regulates the expression of antioxidant scavengers and components of respiratory chain, both are critical for cell survival and homeostasis. Our Preliminary studies also suggest a role of Pitx2 in myofibroblast activity and fibrosis formation. Therefore, targeting Pitx2 for therapeutics provides alternative strategies which focus on cell survival and removing fibrosis. However, an in-depth study of Pitx2 is needed for designing an efficient targeting strategy. The Specific Aim 1 will test the hypothesis that overexpression of Pitx2 promotes the degradation of Nrf2 by inducing the expression of E3 ubiquitin-protein ligase Rbx1. This proposed negative feedback loop may promote the degradation of Nrf2, a key factor for the nuclear translocation and activity of Pitx2. We aim to explain why Pitx2 overexpression in myocardium can only partially repair the myocardium after MI. Transgenic mice with modified Pitx2 and/or Rbx1 expression will be subjected to MI to examine the hypothesis. We will also test an improved therapeutic strategy by overexpressing Pitx2 and Nrf2 simultaneously in infarcted cardiomyocytes. The Specific Aim 2 will focus on the downstream effects of Pitx2 signaling and test the hypothesis that Pitx2 activity in cardiomyocytes inhibits the transition of cardiac fibroblasts to myofibroblasts after MI. Preliminary data suggest that Pitx2 signaling in cardiomyocyte can affect myofibroblast activity. We proposed an interaction between cardiomyocytes and cardiac fibroblasts, coordinated by Pitx2, that can regulate fibrosis formation after MI. Mouse models with modified Pitx2 expression and in vitro primary cell cultures will be used to examine how cardiomyocyte-derived signaling can regulate fibroblast-to-myofibroblast transition, myofibroblast migration, and ECM deposition.